P1359: TUMOR-SPECIFIC, T-CELL-TARGETING MICRORNA IS ASSOCIATED WITH CART CELL FAILURE IN B CELL MALIGNANCIES
Authors: Karan Chohan, Olivia Sirpilla, R. Leo Sakemura, Michelle Cox, Mehrdad Hefazi, Truc Huynh, James Girsch, Kendall Schick, Claudia Manriquez Roman, Carli M. Stewart, Kun Yun, Ismail Can, Ekene J. Ogbodo, Long Mai, Brooke Kimball, Stephen M. Ansell, N. Nora Bennani, Patrick B. Johnston, Jonas Paludo, Jose C. Villasboas-Bisneto, Arushi Khurana, Urshila Durani, Yucai Wang, Paul J. Hampel, Allison Rosenthal, Javier Munoz, Eider Moreno, Januario E. Castro, Hemant S. Murthy, Mohamed Kharfan Dabaja, Sameer Parikh, Neil Kay, Yi Lin, Elizabeth L. Siegler, Saad S. Kenderian
Journal: HemaSphere (2023)

Topic: 24. Gene therapy, cellular immunotherapy and vaccination - Biology & Translational Research Background: Despite the promising efficacy of anti-CD19 chimeric antigen receptor T-cell therapy (CART19) for the treatment of B-cell malignancies, durable remissions remain limited. Aims: To identify the role of tumor-derived extracellular vesicles (EV) micro-RNA (miRNA) cargo in interfering with CART19 functions to impact clinical outcomes. Methods: We interrogated EV miRNA impact on CART19 using two independent patient cohorts and performed functional experiments to validate our findings. The discovery cohort builds on our findings that leukemic-derived EVs induce a state of CART cell exhaustion (Cox et al. 2021). In this cohort, we utilized EVs from patients with chronic lymphocytic leukemia (CLL), interrogated their miRNA profile, and analyzed how their miRNA targets T-cell activation pathways using normal donor laboratory generated CART19 cells. Using this cohort, we identified a miRNA profile that impacts CART cell functions in preclinical models. Next, to determine the impact on CART cell outcomes in the clinic, we interrogated the miRNA profile from patients with diffuse large B cell lymphoma (DLBCL) treated with commercial third-line CART19 cell therapy (axicabtagene ciloleucel, validation cohort). Here, we compared miRNA cargo between responders (complete remission at 6-month evaluation) and non-responders (stable or progressive disease at one-month evaluation). All EVs were isolated from cryopreserved plasma samples collected prospectively from DLBCL patients treated with CART19 cell therapy and CLL patients on IRB-approved protocols. Results: Utilizing CLL EVs from the discovery cohort, we identified significant enrichment of distinct miRNAs targeting T-cell pathways as compared to controls. This led to our hypothesis that tumor-derived, T-cell-targeting miRNAs contribute to CART cell inhibition. Next, using samples from the validation cohort, we studied the miRNA profile differences between responders and non-responders at multiple time points and overlapped our findings with the identified miRNAs from the discovery cohort. Five miRNAs were identified to be differentially expressed. Of relevance, miR-125a was significantly enriched in EVs of non-responders at baseline and remained elevated compared to responders at follow-up intervals (Fig), as well as in EVs derived from CLL patients. miR-125a targets T-cell activation pathways via tumor necrosis factor (TNF) alpha-induced protein 3 and the NF-kB pathway. These findings implicate miR-125a from tumor-derived EVs in the induction of CART19 dysfunction. To delineate the influence of miR-125a directly on CART19 cells, we next performed functional experiments in vitro using CART19 cells generated from healthy donors. Here, we found that CART19 cells transfected with miR-125a showed a dose-dependent decrease in killing of lymphoma cells as compared to miR-control. CART19 cells displayed significantly higher expression of exhaustion markers, including programmed death (PD)-1 and T-cell immunoglobulin domain and mucin domain (TIM)-3, following exposure to miR-125a compared to miR-control. Summary/Conclusion: Our study demonstrates significant alterations in tumor-derived EV miRNA cargo between DLBLC patient responders and non-responders to CART19 therapy. We identified that miR-125a is 1) elevated in non-responders, 2) associated with tumor-derived EV-induced CART19 dysfunction in preclinical models, and 3) able to decrease CART19 efficacy leading to the development of an exhausted phenotype. These results highlight a novel CART19 failure mechanism induced by tumor-secreted EVs and their miRNA cargo.Keywords: Cellular therapy, Chronic lymphocytic leukemia, CAR-T, Lymphoid malignancy